This invention is concerned with the production of viral antigens by persistently infected cell lines and the use of such antigens for diagnostic and other purposes.
U.S. Pat. No. 3,590,127 (issued: Jun. 29, 1971; inventors: J. T. Bryans et al) is concerned with a process for producing equine viral arthritis vaccine. U.S. Pat. No. 3,520,972 (issued: Jul. 20, 1970; inventors: S. E. Smith et al) is concerned with feline virus Vaccines obtained by propagation and serial passage attenuation of virulent feline viruses in diploid feline embryo tissue cell serial passage subculture strains. U.S. Pat. No. 3,432,595 (issued: Mar. 11, 1969; inventor: L. Kasza) is concerned with a canine melanoma cell line and its propagation and use for producing vaccines. A process for producing an equine infectious anemia vaccine, for use in immunizing horses, from organs of sick horses is described and claimed in U.S. Pat. No. 3,873,690 (issued: Mar. 25, 1975; inventor: J. H. Rand III).
Equine infectious anemia (EIA) is a viral disease that occurs naturally in members of the horse family. It is characterized by a diversity of symptoms and may appear in acute, subacute or chronic forms. It was reported first in Europe in 1843 and subsequently about 1880 as a specific disease in Canada.
Equine infectious anemia virus (EIAV) is a retrovirus belonging to the lentivirus subfamily. It contains two glycoproteins (gp70 and gp45) and four major non-glycosylated proteins (p26, p15. p11 and p9). It can cross-react with other retroviruses; for example, sera of EIAV-infected horses precipitated the p25 protein of a retrovirus (LAV or HTLV-III) and a lentivirus associated with acquired immune deficiency syndrome (AIDS) of man.
To date, usually, in vitro EIAV replication for antigen production has been limited to primary cells or diploid finite cell lines. This hampered antigen production because there were repetitive demands for cell stocks and it was necessary to continually screen primary cells for infectious contaminants.
Consequently, if one succeeds in growing EIAV in tumor cells as partially demonstrated using different techniques by Benton et al. Intervirol. 16, 225-232, (1981), but also succeeds in producing a persistently infected potentially infinite cell line, the above restrictions applying to diploid cells can be eliminated and high yields of viral antigens can be expected since malignant cells generally grow at a higher saturation density than diploid cells.
In order that a permanent and productive infection be of use for continuous and large-scale production of antigens the following key problems have to be addressed:
(1) the infection must be made persistent;
(2) antigen release must be continuous;
(3) the cell line must be capable of an indefinite (preferably infinite) life;
(4) a culture system must be developed which allows the production, harvesting and purification of antigens.
By analogy, the infected cell line should be comparable to hybridoma cells releasing monoclonal antibodies.
We believe that we have now achieved this end and can report the persistent EIA infection in a malignantly-transformed canine thymus cell line, Cf2Th, ATCC CRL 1430 and the continuous release of viral antigens for more than 200 serial passages. Furthermore, preliminary data are presented on the production of viral antigens in roller bottles since the EIAV-infected Cf2Th cell cultures release viral antigens in the supernatant fluid.